The broad objective of this proposal is to identify combinatorial vaccine approaches that will result in effective immunization against breast cancer. Immune tolerance mechanisms (systemic and at the tumor site) provide formidable barriers to effective immunization in cancer patients. HER-2/neu transgenic (neu-N) mice demonstrate immune tolerance to the endogenously expressed rat HER-2/neu (neu) protein. We have successfully used these mice to define systemic tolerance mechanisms and to identify combinatorial vaccine approaches that can overcome them. We recently identified RNEU42o-429 as the immunodominant MHC I epitope in rat neu. RNEU42o^29-specific T cells isolated from vaccinated neu-N mice exhibit lower avidity than T cells isolated from the parental non-tolerized mice (FVB/N). Immune modulating doses of Cyclophosphamide (Cy) in sequence with vaccine in neu-N mice produce RNEU42o-429-specific T cells that have 10-fold higher avidity as compared to T cells from neu-N mice given vaccine alone. The enhanced avidity of the T cells correlates with the mouse's ability to reject neu-expressing mammary tumors. Modulation of additional mechanisms of systemic T cell tolerance together with the modulation of mechanisms of immune tolerance in the tumor's microenvironment in these mice, should lead to the development of new and improved combinatorial immunotherapy strategies for the treatment of breast cancer patients. In aim 1, we will evaluate immune modulating agents that will effectively activate neuspecific CD8+ T cell populations specific for the non-dominant neu epitopes.. In aim 2, we will evaluate immune modulating agents that will effectively activate low avidity neu-specific CD8+ T cell populations specific for the RNEU42o-429 epitope. In aim 3, we will evaluate pathways within the tumor's microenvironment (two new B7 family members) that are barriers to effective tumor eradication in vivo. In aim 4, we will evaluate the frequency of expression of these tolerizing signals by human breast cancer specimens. In aim 5, we will determine the therapeutic value of the most potent immune modulating agent/vaccine combinations in treating spontaneously arising neu-expressing mammary tumors and liver metastases. The most effective combinations identified in aims 1-5, will be tested in patients with breast cancer. However, funding for the clinical trials that arise from this grant will come from other sources.